Molecular genetic studies in monogenic and polygenic human diseases.

The main goal of this study was to determine and characterise the types of mutations in two monogenic human disorders: cystic fibrosis (CF) and Duchenne/Becker muscular dystrophy (DMD, BMD) and the susceptibility allele frequency in a polygenic disease: type I insulin-dependent diabetes mellitus (IDDM). After analysing 220 chromosomes for mutations in the CF (Cystic Fibrosis Transmembrane Conductance Regulator = CFTR) gene, delta F508 mutation was most abundant (41%) and out of the non-delta F508 CF mutations 5% was identified as G542X, G551D, R553X, N1303K and W1282X. The CF haplotype analysis by using linked markers to the CFTR gene revealed that the CF "B" haplotype occurred in 66.7% of patients, and this haplotype was 57.2% in patients carrying the delta F508 mutation. Prenatal genetic diagnosis for CF was performed in 10 fetuses: 3 were affected, 6 were carriers, and 1 without any CF mutation. Fifty % of 66 patients with DMB/BMD muscular dystrophy had one or more exon deletions in the dystrophin gene. Eighty-five % of the deletions occurred at the 3' and 15% at the 5' end of the gene. Out of the three prenatal diagnosis in one case DMD was substantiated. Thirty-six % of 50 patients with IDDM possessed four, 44% three and 20% two susceptibility markers in the HLA-DQA1, -DQB1 region. The onset of the disease correlated with the number of susceptibility alleles.

Heat-tolerant methanotrophic bacteria from the hot water effluent of a natural gas field.

Methanotrophic bacteria were isolated from a natural environment potentially favorable to heat-tolerant methanotrophs. An improved colony plate assay was developed and used to identify putative methanotrophic colonies with high confidence. Fourteen new isolates were purified and partially characterized. These new isolates exhibit a DNA sequence homology of up to 97% with the conserved regions in the mmoX and mmoC genes of the soluble methane monooxygenase (MMO)-coding gene cluster of Methylococcus capsulatus Bath. The copper regulation of soluble MMO expression in the same isolates, however, differs from that of M. capsulatus Bath, as the new isolates can tolerate up to 0.8 microM copper without loss of MMO activity while a drastic reduction of MMO activity occurs already at 0.1 microM copper in M. capsulatus Bath. The isolates can be cultivated and utilized at elevated temperatures, and their copper- and heat-tolerant MMO activity makes these bacteria ideal candidates for future biotechnological use.

Development of ependyma in neural transplants.

Under in situ conditions, the innermost (juxtaventricular) neuroepithelial layer of the embryonic brain wall develops into ependyma. No development of ependyma was usually observed, however, in transplanted embryonic brain wall. In our telencephalic transplants, however, cysts lined by epithelium resembling ependyma were observed, although only sporadically. We supposed that occasional foldings of the transplanted telencephalic wall enclosed the aforementioned cysts and so induced the formation of ependyma. This hypothesis was supported by the observation that ependyma developed frequently in a model system in which the telencephalic wall was folded artificially prior to transplantation.

Effect of phytohaemagglutinin on CD45 in T cells.

In this study the effect of PHA activation on the phosphatase activity of CD45 has been investigated in human leukemic T-cell lines. It has been found that in vivo activation of the cells with PHA resulted in 2-4-fold increase in enzyme activity. Addition of PHA to the postnuclear supernatant of cell lysates also resulted in elevation of phosphatase activity. Elevation of enzyme activity resulted from an increase in the amount of antigen in the immunoprecipitates. Elevation of the quantity was not the result of a de novo protein synthesis since the presence of cycloheximide, a protein synthesis inhibitor, did not modulate the effect of PHA. The effect of PHA was specific since ConA, that also bound to the CD45 molecules, or crosslinking of the antigen by antibody did not affect CD45. Since direct binding of PHA to CD45 molecules was shown in immunoblotting analysis, we suggest that the effect of PHA is a consequence of a PHA-induced conformational change of CD45 that results in up-regulation of the analyzed CD45 epitopes.

Vasoactive intestinal polypeptide immunoreactive structures in the mouse barrel field.

Immunohistochemistry for vasoactive intestinal polypeptide was carried out in tangentially cut vibratome sections of the barrel cortex in adult mice. Sections through layer IV have revealed an association between the cytoarchitectonically visible modular organization of barrels and the distribution of immunoreactive axon terminals. These terminals are preferentially localized in the side region of a barrel, whereas the hollow shows a relative scarcity of these structures as shown with image analysis. This finding is the first direct demonstration of a modular distribution of vasoactive intestinal polypeptide-containing axon terminals in the neocortex.

Allele frequencies of cystic fibrosis-linked markers and F508 deletion in affected Hungarian families.

Linked marker haplotype analysis of 16 cystic fibrosis (CF)-affected children, 3 fetuses, 1 healthy child and their parents was performed by restriction fragment length polymorphism (RFLP) for J3.11, Met H, Met D, XV-2c, KM.19 markers. Polymerase chain reaction (PCR) to detect the main mutation of CF chromosome, a specific 3 base pair (bp) deletion (delta F508) was also performed in 17 CF patients. Allelic frequencies on analysed CF chromosomes were: J3.11/Taq I 1.0, 0.0, J3.11/Msp I 0.44, 0.56, Met H/Taq I 0.73, 0.27, Met H/Msp I 0.80, 0.20, Met D/Taq I 0.75, 0.25, XV-2c/Taq I 0.85, 0.15, KM.19/Pst I 0.17, 0.83 for allele 1 and 2, respectively. Two markers, Met H and KM.19 were found to be in strong association with the CF mutation. The frequency of the delta F508 mutation on all 34 CF chromosomes was 0.65 (of which 0.73 was homozygous and 0.27 heterozygous for this deletion).

Increasing pharmacy productivity by expanding the role of pharmacy technicians.

Efforts to meet growing clinical and distributive demands without increasing pharmacy staff are described. Real and expected increases in demands for services led pharmacists at a cancer center to seek ways of accommodating those demands within budgetary limits. Growth in the distributive workload was interfering with clinical consultation work. Research studies by the medical staff were resulting in complex dosage calculations and time-consuming compounding. Increasing requests for clinical services had to be met without compromising distributive services and teaching responsibilities and without raising costs. A plan of action was approved that included the use of a written test and a training manual to allow the hiring and retaining of skilled pharmacy technicians qualified to assume greater responsibilities. Technicians were assigned to enter drug orders into the computer, check other technicians, and dispense certain drugs. Greater use was made of commercially prepared i.v. solutions, and the floor stock was expanded. A comprehensive quality control program was concurrently put in place. The larger role for technicians not only enabled the pharmacy department to increase its distributive workload dramatically but reduced pharmacy medication errors and provided more time for clinical pharmacy practice. The number of pharmacist and technician full-time equivalents increased by only 1.5 in each category between 1985 and 1990. By making more use of pharmacy technicians, a pharmacy department was able to meet escalating demands for services with only a minor increase in personnel.

Developmental gradients of vasoactive intestinal polypeptide (VIP)-containing neurons in the rat visual cortex detected by image analysis.

The postnatal development of vasoactive intestinal polypeptide-immunoreactive (VIP-IR) neurons was followed by computer-assisted image analysis in the rat visual cortex. The laminar distribution of all VIP-IR structures was measured. These structures were subdivided into cell somata, dendritic profiles and axonal boutons and measured separately. VIP-IR neurons were first seen on postnatal day 1, mostly in the upper half of the presumptive visual cortex. A localization of cell bodies similar to that in the adult is reached between days 12 and 16. VIP-IR dendrites have a protracted growth period as compared to perikarya, involving a developmental gradient from an even distribution to a concentration in the upper cortical layers. This is due to the formation of dendritic terminal arbors after the second postnatal week. Scattered VIP-IT axonal boutons appear on day 3 in the midportion of the presumptive visual cortex. Their typical laminar distribution in layers II, IV and lower VI was observed after day 12. Our results suggest that the biochemically detected sharp increase in VIP levels after the second postnatal week is due to the maturation of cell processes as a morphological basis of neuronal connectivity.

Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide.

Synthetic genes coding for elongated precursors of the vasoactive intestinal polypeptide (VIP) were synthesized and cloned in a highly efficient Escherichia coli expression vector. The synthetic genes code for VIP with its methionine (at position 17) replaced by leucine and elongated at the C-terminus by Gly (vasoactive intestinal polypeptide-Gly, i.e. VIPa) or by Gly-Lys-Arg (vasoactive intestinal polypeptide-Gly-Lys-Arg, i.e. VIPb). The synthetic genes fused to the N-terminal part of the E. coli beta-galactosidase gene were expressed to yield high amounts of fusion proteins reaching upon induction at least 60% of the total cellular protein. The fusion proteins of 314 and 316 amino acids carrying in their C-terminal portion either the 29 or 31 amino acids long VIP precursor polypeptide were shown to be immunoreactive with VIP antisera and were further purified and cleaved by CNBr. The resulting purified peptide precursors (VIPa and VIPb) were recognized by VIP receptors in rat liver plasma membranes and by antibodies to porcine VIP in a radioimmunoassay. Both precursors activated adenylate cyclase in rat liver membranes and stimulated pancreatic secretion in the cat. The affinity and potency of the cloned precursors is close to that of VIP purified from porcine intestine, suggesting that the elongated VIP precursors may act even without being converted into the C-terminal amide form of the peptide. The elongated VIP precursors expressed in E. coli may provide a cheap, large-scale source of experimental material for studies on VIP actions.

Comparative studies on fresh and postmortem isolated synaptosomes: ATPase activities and ouabain binding.

In earlier studies we have established that rat cortical synaptosomes isolated after a 2-h postmortem storage period showed functional performance and morphological characteristics similar to those isolated immediately after decapitation. The present paper is an account of further functional studies involving the activity of Na-K- and Mg-dependent ATPase and the binding of ouabain in postmortem synaptosomes. These functions were found to survive without major alteration even in the 6-h postmortem fractions. On the other hand the 6-h postmortem fractions showed a very slow oxygen consumption unaffected by DNP and veratrine. In both control and postmortem synaptosomes the actual activity of Na-K-ATPase as calculated from the ouabain-sensitive component of oxygen consumption was consistently less than the potential activity of the enzyme revealed if determination was carried out on the basis of phosphate splitting.

Types and spatial distribution of vasoactive intestinal polypeptide (VIP)-containing synapses in the rat visual cortex.

In the rat visual cortex vasoactive intestinal polypeptide (VIP)-containing structures were studied by means of light and electron microscopy and image analysis. VIP-immunoreactive axon terminals were found to form symmetric synapses with small dendritic shafts, dendritic spines and somata of pyramidal cells and interneurons. VIP-terminals often occurred in pairs with VIP-negative, asymmetric synapses on the same postsynaptic structure. VIP-immunostained dendrites and perikarya were contacted by a purely asymmetric and a mixed population of VIP-negative terminals, respectively. Synaptic connections between two VIP-neurons are seldom as compared to the other types of VIP-synapses. Quantitative studies obtained by the image analysis of VIP-stained boutons and dendritic particles in light microscopic preparations suggest a distinct laminar distribution. Dendritic particles are most frequent in layers I-II, whereas axonal boutons have three laminar accumulations: at the border of layers I-II, in layer IV and layer VI. Together with previous results, the present findings argue for a non-random spatial distribution of VIP-boutons.

Effect of calcium on Na-K-ATPase activity in depolarized rat cortical synaptosomes as determined on the basis of oxygen consumption.

The oxygen consumption of rat cortical synaptosomes together with its ouabain-sensitive component were measured after veratrine-induced depolarization in the presence and in the absence of calcium, and with calcium added after depolarization by veratrine or potassium. Taking the ouabain-sensitive component of oxygen consumption as an indicator of Na-K-ATPase activity it was concluded that calcium entry during depolarization decreased synaptosomal Na-K-ATPase activity only slightly and in a protracted manner. Consequently, Ca++-induced inhibition of Na-K-ATPase does not seem to serve as a "trigger" mechanism of transmitter release.

The ouabain-sensitive component of synaptosomal respiration.

The relationship of oxygen consumption with the activity of the synaptic Na-K-pump was investigated in rat cortical synaptosomes. Changes in oxygen consumption were monitored during the restitution of physiological ionic equilibrium in incubated synaptosomes and under the effects of ouabain and protoveratrine. The ouabain-sensitive component of synaptosomal respiration was measured and correlated with earlier data on synaptosomal K+-uptake. This ouabain-sensitive component was shown to reflect the activity of the Na-K-pump. Our results thus provide a means for the continuous monitoring of ion-pump activity in intact synaptosomes.

The effect of pentobarbital, chloralhydrate, ether and protoveratrine on the distribution of synaptic vesicles in rat cortical synaptosomes.

Rat cortical synaptosomes were incubated in a saline medium. On the addition of pentobarbital, chloralhydrate, ether and protoveratrine a redistribution of synaptic vesicles was revealed by electron microscopy. The anaesthetics decreased the number of synaptic vesicles attached to the presynaptic membrane while protoveratrine increased them. It is assumed that these agents affect the mechanism of vesicle reformation from the synaptosomal membrane and/or vesicle migration towards the synaptic cleft.

Radiologic soft tissue shadows in the pelvis: another look.

In patients with adequate amounts of intramuscular fat, the normal contours of the muscles of the pelvic floor and side-walls may be visualized on plain radiographs. Knowledge of the relationships of these muscles to surrounding structures has been facilitated by computed tomography. Obliteration of the medial margin of the psoas muscle with an attendant soft tissue mass implies pathology in the region of the medial chain of external iliac lymph nodes; similarly, obliteration of the obturator internus shadow suggests internal iliac adenopathy. Nonnodal masses in these regions can cause similar radiographic abnormalities. The normal shadow of the piriformis muscle may simulate external iliac adenopathy. Pitfalls which may lead to overinterpretation are discussed.

Whole body computed tomography in the diagnosis of abdominal and thoracic malignancy: review of 600 cases.

Whole-body computed tomography (CT) was performed on 600 patients with known or suspected malignancy at the National Institutes of Health. This new diagnostic modality utilizes a finely columnated X-ray source mounted opposite a computer-linked detector bank to generate anatomic cross-sectional images of the body part being scanned. CT proved valuable in defining many pathologic processes and was, at times, deemed the only test short of exploratory surgery that yielded diagnostic information. This latter benefit was most acutely emphasized in lesions affecting the retroperitoneum, adrenal glands, pelvis, liver, and immediate subpleural pulmonary parenchyma.